Difference between revisions of "Part:BBa K4201020:Design"

(→‎References)
(→‎Design Notes)
 
(4 intermediate revisions by the same user not shown)
Line 9: Line 9:
 
Part information and design consideration can be found on respective basic parts pages.
 
Part information and design consideration can be found on respective basic parts pages.
  
 +
In contrast to other composite parts designed by our group, each individual gene in this assembly has its own promoter and terminator, which allows us to study and compare which combinations of adjoining or separated genes results in the best expression of our target genes.
  
 +
Gmubi is constitutive promoter native to <i>Glyine max</i><sup>5</sup>, while AtHSP is the terminator of a heat shock protein that has shown to promote expression in plants<sup>6</sup>.
 +
 +
This assembly was made using NEB 10-<i>beta</i> cells and GoldBio EHA105 <i>agrobacterium</i> as intermediate hosts. Parts BBa_K4201013 (Amp BsaI) and BBa_K4201014 (Chlor AaRI) were used as intermediate backbones for Golden Gate assembly. The construct was integrated into part BBa_K4201015 (Kan BsaI) as a final backbone before transfection into <i>Glycine max</i>.
  
 
===Source===
 
===Source===
  
−
CrtE is a GGPP synthase from <i> Pantoea ananatis LMG 20103 </i>  
+
CrtE is a GGPP synthase from <i> Pantoea ananatis LMG 20103</i><sup>1</sup>.
  
−
RUBY is a reporter gene from the order Caryophyllales.  
+
Gmubi promoters originate from <i>Glycine max</i><sup>5</sup>.
  
−
Gmubi promoters originate from <i>Glycine max</i>.
+
AtHSP terminators originate from the <i>Arabidopsis thaliana</i> genome<sup>6</sup>.  
  
−
AtHSP terminators originate from the <i>Arabidopsis thaliana</i> genome.  
+
cytoTDS2 is a taxadiene synthase native to <i>Taxus chinensis var. mairei</i> optimized for use in <i>Glycine max</i><sup>2</sup>.
  
−
cytoTDS2 is a taxadiene synthase native to <i>Taxus chinensis var. mairei</i> optimized for use in <i>Glycine max</i>.
+
T5αOH is a hydroxylase from <i>Taxus baccata</i> that converts taxadiene into taxadiene-5α-ol, a step in the paclitaxel pathway<sup>3</sup>.  
  
−
T5αOH is a hydroxylase from <i>Taxus baccata</i> that converts taxadiene into taxadiene-5α-ol, a step in the paclitaxel pathway.  
+
RUBY is a reporter gene from the order Caryophyllales<sup>4</sup>.
  
  
 
<i>de novo</i> Synthesis was completed by iGem sponsors IDT and Twist Biosciences.
 
<i>de novo</i> Synthesis was completed by iGem sponsors IDT and Twist Biosciences.
 +
 +
===References===
 +
1. Majer, E., Llorente, B., Rodríguez-Concepción, M. & Daròs, J.-A. Rewiring carotenoid biosynthesis in plants using a viral vector. Sci. Rep. 7, 41645 (2017).
 +
 +
2. Xiong, X. et al. The Taxus genome provides insights into paclitaxel biosynthesis. Nat. Plants 7, 1026–1036 (2021).
 +
 +
3. Edgar, S. et al. Mechanistic Insights into Taxadiene Epoxidation by Taxadiene-5α-Hydroxylase. ACS Chem. Biol. 11, 460–469 (2016).
 +
 +
4. He, Y., Zhang, T., Sun, H., Zhan, H. & Zhao, Y. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic. Res. 7, 1–6 (2020).
 +
 +
5. De La Torre, C. M. & Finer, J. J. The intron and 5’ distal region of the soybean Gmubi promoter contribute to very high levels of gene expression in transiently and stably transformed tissues. Plant Cell Rep. 34, 111–120 (2015).
 +
 +
6. Nagaya, S., Kawamura, K., Shinmyo, A. & Kato, K. The HSP Terminator of Arabidopsis thaliana Increases Gene Expression in Plant Cells. Plant Cell Physiol. 51, 328–32 (2010).

Latest revision as of 09:58, 12 October 2022


CrtE_cytoTDS2__T5αOH_RUBY


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 6848
    Illegal PstI site found at 2087
    Illegal PstI site found at 5638
    Illegal PstI site found at 8395
    Illegal PstI site found at 13603
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 6848
    Illegal PstI site found at 2087
    Illegal PstI site found at 5638
    Illegal PstI site found at 8395
    Illegal PstI site found at 13603
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 6848
    Illegal BglII site found at 790
    Illegal BglII site found at 1153
    Illegal BglII site found at 2021
    Illegal BglII site found at 2956
    Illegal BglII site found at 4011
    Illegal BglII site found at 5324
    Illegal BglII site found at 5572
    Illegal BamHI site found at 11815
    Illegal XhoI site found at 3601
    Illegal XhoI site found at 3868
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 6848
    Illegal PstI site found at 2087
    Illegal PstI site found at 5638
    Illegal PstI site found at 8395
    Illegal PstI site found at 13603
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 6848
    Illegal PstI site found at 2087
    Illegal PstI site found at 5638
    Illegal PstI site found at 8395
    Illegal PstI site found at 13603
    Illegal NgoMIV site found at 1697
    Illegal NgoMIV site found at 6997
    Illegal NgoMIV site found at 9751
    Illegal NgoMIV site found at 10330
    Illegal NgoMIV site found at 12043
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI site found at 948
    Illegal BsaI site found at 1887
    Illegal BsaI site found at 2167
    Illegal BsaI site found at 3113
    Illegal BsaI site found at 5438
    Illegal BsaI site found at 5718
    Illegal BsaI.rc site found at 934
    Illegal BsaI.rc site found at 1873
    Illegal BsaI.rc site found at 2153
    Illegal BsaI.rc site found at 3100
    Illegal BsaI.rc site found at 5424
    Illegal BsaI.rc site found at 5704
    Illegal BsaI.rc site found at 6651


Design Notes

Part information and design consideration can be found on respective basic parts pages.

In contrast to other composite parts designed by our group, each individual gene in this assembly has its own promoter and terminator, which allows us to study and compare which combinations of adjoining or separated genes results in the best expression of our target genes.

Gmubi is constitutive promoter native to Glyine max5, while AtHSP is the terminator of a heat shock protein that has shown to promote expression in plants6.

This assembly was made using NEB 10-beta cells and GoldBio EHA105 agrobacterium as intermediate hosts. Parts BBa_K4201013 (Amp BsaI) and BBa_K4201014 (Chlor AaRI) were used as intermediate backbones for Golden Gate assembly. The construct was integrated into part BBa_K4201015 (Kan BsaI) as a final backbone before transfection into Glycine max.

Source

CrtE is a GGPP synthase from Pantoea ananatis LMG 201031.

Gmubi promoters originate from Glycine max5.

AtHSP terminators originate from the Arabidopsis thaliana genome6.

cytoTDS2 is a taxadiene synthase native to Taxus chinensis var. mairei optimized for use in Glycine max2.

T5αOH is a hydroxylase from Taxus baccata that converts taxadiene into taxadiene-5α-ol, a step in the paclitaxel pathway3.

RUBY is a reporter gene from the order Caryophyllales4.


de novo Synthesis was completed by iGem sponsors IDT and Twist Biosciences.

References

1. Majer, E., Llorente, B., Rodríguez-Concepción, M. & Daròs, J.-A. Rewiring carotenoid biosynthesis in plants using a viral vector. Sci. Rep. 7, 41645 (2017).

2. Xiong, X. et al. The Taxus genome provides insights into paclitaxel biosynthesis. Nat. Plants 7, 1026–1036 (2021).

3. Edgar, S. et al. Mechanistic Insights into Taxadiene Epoxidation by Taxadiene-5α-Hydroxylase. ACS Chem. Biol. 11, 460–469 (2016).

4. He, Y., Zhang, T., Sun, H., Zhan, H. & Zhao, Y. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic. Res. 7, 1–6 (2020).

5. De La Torre, C. M. & Finer, J. J. The intron and 5’ distal region of the soybean Gmubi promoter contribute to very high levels of gene expression in transiently and stably transformed tissues. Plant Cell Rep. 34, 111–120 (2015).

6. Nagaya, S., Kawamura, K., Shinmyo, A. & Kato, K. The HSP Terminator of Arabidopsis thaliana Increases Gene Expression in Plant Cells. Plant Cell Physiol. 51, 328–32 (2010).